Divergence in Reproductive Behaviors Is Associated with the Evolutionary Loss of Parental Care.

AbstractThe mechanisms underlying the divergence of reproductive strategies between closely related species are still poorly understood. Additionally, it is unclear which selective factors drive the evolution of reproductive behavioral variation and how these traits coevolve, particularly during early divergence. To address these questions, we quantified behavioral differences in a recently diverged pair of Nova Scotian three-spined stickleback (Gasterosteus aculeatus) populations, which vary in parental care, with one population displaying paternal care and the other lacking this. We compared both populations, and a full reciprocal F1 hybrid cross, across four major reproductive stages: territoriality, nesting, courtship, and parenting. We identified significant divergence in a suite of heritable behaviors. Importantly, F1 hybrids exhibited a mix of behavioral patterns, some of which suggest sex linkage. This system offers fresh insights into the coevolutionary dynamics of reproductive behaviors during early divergence and offers support for the hypothesis that coevolutionary feedback between sexual selection and parental care can drive rapid evolution of reproductive strategies.

Does the loss of diadromy imply the loss of salinity tolerance? A gene expression study with replicate nondiadromous populations of Galaxias maculatus.

The recurrent colonization of freshwater habitats and subsequent loss of diadromy is a major ecological transition that has been reported in many ancestrally diadromous fishes. Such residency is often accompanied by a loss of tolerance to seawater. The amphidromous Galaxias maculatus has repeatedly colonized freshwater streams with evidence that freshwater-resident populations exhibit stark differences in their tolerance to higher salinities. Here, we used transcriptomics to gain insight into the mechanisms contributing to reduced tolerance to higher salinities in freshwater resident populations. We conducted an acute salinity challenge (0 ppt to 23-25 ppt) and measured osmoregulatory ability (muscle water content) over 48 h in three populations: diadromous, saltwater intolerant resident (Toltén), and saltwater tolerant resident (Valdivia). RNA sequencing of the gills identified genes that were differentially expressed in association with the salinity change and associated with the loss of saltwater tolerance in the Toltén population. Key genes associated with saltwater acclimation were characterized in diadromous G. maculatus individuals, some of which were also expressed in the saltwater tolerant resident population (Valdivia). We found that some of these "saltwater acclimation" genes, including the cystic fibrosis transmembrane conductance regulator gene (CFTR), were not significantly upregulated in the saltwater intolerant resident population (Toltén), suggesting a potential mechanism for the loss of tolerance to higher salinities. As the suite of differentially expressed genes in the diadromous-resident comparison differed between freshwater populations, we hypothesize that diadromy loss results in unique evolutionary trajectories due to drift, so the loss of diadromy does not necessarily lead to a loss in upper salinity tolerance.

La colonización recurrente de hábitats de agua dulce y la subsecuente pérdida de diadromía es una transición ecológica importante que ha sido reportada en varias especies de peces con ancestros diádromos. Esta residencia está acompañada frecuentemente por la pérdida de tolerancia a ambientes de agua salada. Galaxias maculatus, especie anfídroma, ha colonizado ríos repetidamente y existe evidencia que las poblaciones residentes presentan diferencias respecto a la tolerancia al agua salada. En este estudio, usamos transcriptómica para dilucidar los mecanismos que contribuyen a la reducida tolerancia a altas salinidades en las poblaciones residentes de agua dulce. Realizamos un desafío agudo de salinidad (0 ppt a 23-2 ppt) y medimos la habilidad osmoreguladora (contenido de agua en músculo) por 48 horas en individuos de tres poblaciones: una diádroma, una intolerante a agua salada (Toltén) y una tolerante a agua salada (Valdivia). Con el secuenciamiento de ARN de las branquias identificamos los genes expresados diferencialmente al cambio de salinidad y cuales están asociados a la pérdida de tolerancia a agua salada en la población de Toltén. Genes claves asociados a la aclimatación al agua salada fueron caracterizados en individuos diádromos, algunos de ellos también se expresaron en la población residente tolerante al agua salada (Valdivia). Sin embargo, algunos genes involucrados en la aclimatación al agua salada, incluyendo el gen regulador de la conductancia transmembrana de la fibrosis quística (CFTR), no se diferenciaron significativamente en la población residente intolerante al agua salada (Toltén), sugiriendo un mecanismo potencial de la pérdida de tolerancia a ambientes con salinidad elevada. Como el conjunto de genes expresados difiere entre las dos poblaciones residentes al compararse con la población diádroma, hipotetizamos que la pérdida de diadromía resulta en trayectorias evolutivas únicas debido a deriva génica, por lo que la pérdida de la diadromía no necesariamente conlleva a la pérdida de la tolerancia a aguas saladas.

Reproductive isolating mechanisms contributing to asymmetric hybridization in Killifishes (Fundulus spp.).

When species hybridize, one F1 hybrid cross type often predominates. Such asymmetry can arise from differences in a variety of reproductive barriers, but the relative roles and concordance of pre-mating, post-mating prezygotic, and post-zygotic barriers in producing these biases in natural animal populations have not been widely investigated. Here, we study a population of predominantly F1 hybrids between two killifish species (Fundulus heteroclitus and F. diaphanus) in which >95% of F1 hybrids have F. diaphanus mothers and F. heteroclitus fathers (D♀ × H♂). To determine why F. heteroclitus × F. diaphanus F1 hybrids (H♀ × D♂) are so rare, we tested for asymmetry in pre-mating reproductive barriers (female preference and male aggression) at a common salinity (10 ppt) and post-mating, pre-zygotic (fertilization success) and post-zygotic (embryonic development time and hatching success) reproductive barriers at a range of ecologically relevant salinities (0, 5, 10, and 15 ppt). We found that F. heteroclitus females preferred conspecific males, whereas F. diaphanus females did not, matching the observed cross bias in the wild. Naturally rare H♀ × D♂ crosses also had lower fertilization success than all other cross types, and a lower hatching success than the prevalent D♀ × H♂ crosses at the salinity found in the hybrid zone centre (10 ppt). Furthermore, the naturally predominant D♀ × H♂ crosses had a higher hatching success than F. diaphanus crosses at 10 ppt, which may further increase their relative abundance. The present study suggests that a combination of incomplete mating, post-mating pre-zygotic and post-zygotic reproductive isolating mechanisms act in concert to produce hybrid asymmetry in this system.

Parental early life environments drive transgenerational plasticity of offspring metabolism in a freshwater fish (Danio rerio).

Parental experiences can lead to changes in offspring phenotypes through transgenerational plasticity (TGP). TGP is expected to play a role in improving the responses of offspring to changes in climate, but little is known about how the early lives of parents influence offspring TGP. Here, we use a model organism, zebrafish (Danio rerio), to contrast the effects of early and later life parental thermal environments on offspring routine metabolism. To accomplish this, we exposed both parents to either constant optimal (27°C) or environmentally realistic diel fluctuating (22-32°C) temperatures during early (embryonic and larval) and later (juvenile and adult) life in a factorial design. We found significant reduction of routine metabolic rates (greater than 20%) at stressful temperatures (22°C and 32°C) after biparental early life exposure to fluctuating temperatures, but little effect of later life parental temperatures on offspring metabolism. This reduction reflects metabolic compensation and is expected to enhance offspring body sizes under stressful temperatures. These changes occur over and above the effects of parental environments on egg size, suggesting alternate non-genetic mechanisms influenced offspring metabolic rates.

Heterogeneous Histories of Recombination Suppression on Stickleback Sex Chromosomes.

How consistent are the evolutionary trajectories of sex chromosomes shortly after they form? Insights into the evolution of recombination, differentiation, and degeneration can be provided by comparing closely related species with homologous sex chromosomes. The sex chromosomes of the threespine stickleback (Gasterosteus aculeatus) and its sister species, the Japan Sea stickleback (G. nipponicus), have been well characterized. Little is known, however, about the sex chromosomes of their congener, the blackspotted stickleback (G. wheatlandi). We used pedigrees to obtain experimentally phased whole genome sequences from blackspotted stickleback X and Y chromosomes. Using multispecies gene trees and analysis of shared duplications, we demonstrate that Chromosome 19 is the ancestral sex chromosome and that its oldest stratum evolved in the common ancestor of the genus. After the blackspotted lineage diverged, its sex chromosomes experienced independent and more extensive recombination suppression, greater X-Y differentiation, and a much higher rate of Y degeneration than the other two species. These patterns may result from a smaller effective population size in the blackspotted stickleback. A recent fusion between the ancestral blackspotted stickleback Y chromosome and Chromosome 12, which produced a neo-X and neo-Y, may have been favored by the very small size of the recombining region on the ancestral sex chromosome. We identify six strata on the ancestral and neo-sex chromosomes where recombination between the X and Y ceased at different times. These results confirm that sex chromosomes can evolve large differences within and between species over short evolutionary timescales.

Alternative splicing: An overlooked mechanism contributing to local adaptation?

Identifying the molecular mechanisms contributing to phenotypic variation in natural populations is a major goal of molecular ecology. However, the multiple regulatory steps between genotype and phenotype mean that many potential mechanisms can lead to trait divergence. To date, the role of transcriptional regulation in local adaptation has received much focus, as we can readily measure mRNA quantity and have a reasonable grasp of how variation in the expression of many protein-coding genes can influence phenotype. Thus, studying the evolution of protein-coding gene mRNA abundance in candidate tissues has led to successes in detecting the molecular mechanisms underlying local adaptation (reviewed by Hill et al., 2021). However, the contribution of differential splicing of precursor mRNA (pre-mRNA) to adaptive differentiation, as well as the loci controlling this variation, remains largely unexplored in wild populations. In their "From the Cover'" article in this issue of Molecular Ecology, Jacobs and Elmer (2021) reanalyse muscle RNA sequencing (RNA-seq) data to quantify the relative contributions of variation in mRNA quantity (differentially expressed "DE" genes) and splice variant identity (differentially spliced "DS" genes) to parallel divergence of wild "benthic" and "pelagic" ecotypes of a salmonid fish, the Arctic charr (Salvelinus alpinus). They found little overlap in the identity and biological functions of DE and DS genes, suggesting that these two regulatory mechanisms act on different cellular traits to complementarily alter organismal phenotype. Furthermore, many DE and DS genes could be mapped to cis-acting QTL, arguing that some of this regulatory divergence is genetically based. DE and DS genes were also more likely to be "hub genes" than their nondivergent counterparts, hinting that this regulatory variation may have a variety of phenotypic effects. The comparison of three independently evolved pairs of benthic and pelagic charr uncovered greater than expected parallelism in both expression and splicing between ecotypes across different lakes, supporting a role for these molecular phenotypes in adaptive divergence. Overall, the findings of Jacobs and Elmer (2021) highlight the importance of alternative splicing as a potential mechanism underlying local adaptation and provide a framework for others hoping to make the most of their RNA-seq data.

Physiological insight into the evolution of complex phenotypes: aerobic performance and the O2 transport pathway of vertebrates.

Evolutionary physiology strives to understand how the function and integration of physiological systems influence the way in which organisms evolve. Studies of the O2 transport pathway - the integrated physiological system that transports O2 from the environment to mitochondria - are well suited to this endeavour. We consider the mechanistic underpinnings across the O2 pathway for the evolution of aerobic capacity, focusing on studies of artificial selection and naturally selected divergence among wild populations of mammals and fish. We show that evolved changes in aerobic capacity do not require concerted changes across the O2 pathway and can arise quickly from changes in one or a subset of pathway steps. Population divergence in aerobic capacity can be associated with the evolution of plasticity in response to environmental variation or activity. In some cases, initial evolutionary divergence of aerobic capacity arose exclusively from increased capacities for O2 diffusion and/or utilization in active O2-consuming tissues (muscle), which may often constitute first steps in adaptation. However, continued selection leading to greater divergence in aerobic capacity is often associated with increased capacities for circulatory and pulmonary O2 transport. Increases in tissue O2 diffusing capacity may augment the adaptive benefit of increasing circulatory O2 transport owing to their interactive influence on tissue O2 extraction. Theoretical modelling of the O2 pathway suggests that O2 pathway steps with a disproportionately large influence over aerobic capacity have been more likely to evolve, but more work is needed to appreciate the extent to which such physiological principles can predict evolutionary outcomes.

Convergence in organ size but not energy metabolism enzyme activities among wild Lake Whitefish (Coregonus clupeaformis) species pairs.

The repeated evolution of similar phenotypes by similar mechanisms can be indicative of local adaptation, constraints or biases in the evolutionary process. Little is known about the incidence of physiological convergence in natural populations, so here we test whether energy metabolism in 'dwarf' and 'normal' Lake Whitefish evolves by similar mechanisms. Prior genomic and transcriptomic studies have found that divergence in energy metabolism is key to local adaptation in whitefish species pairs, but that distinct genetic and transcriptomic changes often underlie phenotypic evolution among lakes. Here, we predicted that traits at higher levels of biological organization, including the activities of energy metabolism enzymes (the product of enzyme concentration and turnover rate) and the relative proportions of metabolically active tissues (heart, liver, skeletal muscle), would show greater convergence than genetic and transcriptomic variation. We compared four whitefish species pairs and found convergence in organ size whereby all dwarf whitefish populations have a higher proportion of red skeletal muscle, three have relatively larger livers and two have relatively larger ventricles than normal fish. On the other hand, hepatic and muscle enzyme activities showed little convergence and were largely dependent on lake of origin. Only the most genetically divergent species pair (Cliff Lake) displayed white muscle enzyme activities matching results from laboratory-reared normal and dwarf whitefish. Overall, these data show convergence in the evolution of organ size, but not in the activities of candidate enzymes of energy metabolism, which may have evolved mainly as a consequence of demographic or ecological differences among lakes.

Adaptation and acclimation of traits associated with swimming capacity in Lake Whitefish (coregonus clupeaformis) ecotypes.

BACKGROUND: Improved performance in a given ecological niche can occur through local adaptation, phenotypic plasticity, or a combination of these mechanisms. Evaluating the relative importance of these two mechanisms is needed to better understand the cause of intra specific polymorphism. In this study, we reared populations of Lake Whitefish (Coregonus clupeaformis) representing the'normal' (benthic form) and the 'dwarf' (derived limnetic form) ecotypes in two different conditions (control and swim-training) to test the relative importance of adaptation and acclimation in the differentiation of traits related to swimming capacity. The dwarf whitefish is a more active swimmer than the normal ecotype, and also has a higher capacity for aerobic energy production in the swimming musculature. We hypothesized that dwarf fish would show changes in morphological and physiological traits consistent with reductions in the energetic costs of swimming and maintenance metabolism. RESULTS: We found differences in traits predicted to decrease the costs of prolonged swimming and standard metabolic rate and allow for a more active lifestyle in dwarf whitefish. Dwarf whitefish evolved a more streamlined body shape, predicted to lead to a decreased drag, and a smaller brain, which may decrease their standard metabolic rate. Contrary to predictions, we also found evidence of acclimation in liver size and metabolic enzyme activities. CONCLUSION: Results support the view that local adaptation has contributed to the genetically-based divergence of traits associated with swimming activity. Presence of post-zygotic barriers limiting gene flow between these ecotype pairs may have favoured repeated local adaptation to the limnetic niches.

Origins and functional diversification of salinity-responsive Na(+) , K(+) ATPase α1 paralogs in salmonids.

The Salmoniform whole-genome duplication is hypothesized to have facilitated the evolution of anadromy, but little is known about the contribution of paralogs from this event to the physiological performance traits required for anadromy, such as salinity tolerance. Here, we determined when two candidate, salinity-responsive paralogs of the Na(+) , K(+) ATPase α subunit (α1a and α1b) evolved and studied their evolutionary trajectories and tissue-specific expression patterns. We found that these paralogs arose during a small-scale duplication event prior to the Salmoniform, but after the teleost, whole-genome duplication. The 'freshwater paralog' (α1a) is primarily expressed in the gills of Salmoniformes and an unduplicated freshwater sister species (Esox lucius) and experienced positive selection in the freshwater ancestor of Salmoniformes and Esociformes. Contrary to our predictions, the 'saltwater paralog' (α1b), which is more widely expressed than α1a, did not experience positive selection during the evolution of anadromy in the Coregoninae and Salmonine. To determine whether parallel mutations in Na(+) , K(+) ATPase α1 may contribute to salinity tolerance in other fishes, we studied independently evolved salinity-responsive Na(+) , K(+) ATPase α1 paralogs in Anabas testudineus and Oreochromis mossambicus. We found that a quarter of the mutations occurring between salmonid α1a and α1b in functionally important sites also evolved in parallel in at least one of these species. Together, these data argue that paralogs contributing to salinity tolerance evolved prior to the Salmoniform whole-genome duplication and that strong selection and/or functional constraints have led to parallel evolution in salinity-responsive Na(+) , K(+) ATPase α1 paralogs in fishes.

Ecological proteomics: finding molecular markers that matter.

It is becoming increasingly clear that local adaptation can occur even in the face of high gene flow and limited overall genomic differentiation among populations (reviewed by Nosil et al. 2009). Thus, one important task for molecular ecologists is to sift through genomic data to identify the genes that matter for local adaptation (Hoffmann & Willi 2008; Stapley et al. 2010). Recent advances in high-throughput molecular technologies have facilitated this search, and a variety of approaches can be applied, including those grounded in population genetics [e.g. outlier analysis (Pavlidis et al. 2008)], classical and quantitative genetics [e.g. quantitative trait locus analysis (MacKay et al. 2009)], and cellular and molecular biology [e.g. transcriptomics (Larsen et al. 2011)]. However, applying these approaches in nonmodel organisms that lack extensive genetic and genomic resources has been a formidable challenge. In this issue, Papakostas et al. (2012). demonstrate how one such approach ­ high-throughput label-free proteomics (reviewed by Gstaiger & Aebersold 2009; Domon & Aebersold 2010) ­ can be applied to detect genes that may be involved in local adaptation in a species with limited genomic resources. Using this approach, they identified genes that may be implicated in local adaptation to salinity in European whitefish (Coregonus lavaretus L.) and provide insight into the mechanisms by which fish cope with changes in this critically important environmental parameter.

Mechanisms underlying parallel reductions in aerobic capacity in non-migratory threespine stickleback (Gasterosteus aculeatus) populations.

Non-migratory, stream-resident populations of threespine stickleback, Gasterosteus aculeatus, have a lower maximum oxygen consumption ((O(2),max)) than ancestral migratory marine populations. Here, we examined laboratory-bred stream-resident and marine crosses from two locations (West and Bonsall Creeks) to determine which steps in the oxygen transport and utilization cascade evolved in conjunction with, and thus have the potential to contribute to, these differences in (O(2),max). We found that West Creek stream-resident fish have larger muscle fibres (not measured in Bonsall fish), Bonsall Creek stream-resident fish have smaller ventricles, and both stream-resident populations have evolved smaller pectoral adductor and abductor muscles. However, many steps of the oxygen cascade did not evolve in stream-resident populations (gill surface area, hematocrit, mean cellular hemoglobin content and the activities of mitochondrial enzymes per gram ventricle and pectoral muscle), arguing against symmorphosis. We also studied F1 hybrids to determine which traits in the oxygen cascade have a genetic architecture similar to that of (O(2),max). In West Creek, (O(2),max), abductor and adductor size all showed dominance of marine alleles, whereas in Bonsall Creek, (O(2),max) and ventricle mass showed dominance of stream-resident alleles. We also found genetically based differences among marine populations in hematocrit, ventricle mass, pectoral muscle mass and pectoral muscle pyruvate kinase activity. Overall, reductions in pectoral muscle mass evolved in conjunction with reductions in (O(2),max) in both stream-resident populations, but the specific steps in the oxygen cascade that have a genetic basis similar to that of (O(2),max), and are thus predicted to have the largest impact on (O(2),max), differ among populations.

Correlates of prolonged swimming performance in F2 hybrids of migratory and non-migratory threespine stickleback.

Determining which underlying traits contribute to differences in whole-animal performance can be difficult when many traits differ between individuals with high and low capacities. We have previously found that migratory (anadromous marine) and non-migratory (stream-resident) threespine stickleback (Gasterosteus aculeatus) populations have genetically based differences in prolonged swimming performance (U(crit)) that are associated with divergence of a number of candidate morphological and physiological traits (pectoral fin size and shape, body shape, pectoral muscle and heart size, and pectoral muscle metabolic enzyme activities). Here, we use F2 hybrid crosses to determine which traits are correlated with U(crit) when expressed in a largely randomized genetic background and a range of trait values for other divergent traits. We found that four of our 12 candidate traits were positively correlated with U(crit) in F2 hybrids and that the combined effects of ventricle mass, pectoral adductor mass and adductor citrate synthase activity accounted for 17.9% of the variation in U(crit). These data provide additional support for a causal role of muscle and heart size in mediating intraspecific differences in U(crit), but indicate that many candidate morphological and biochemical traits do not have a strong effect on U(crit) when disassociated from other divergent traits. However, the limited variation in U(crit) in our F2 hybrid families may have decreased our ability to detect correlations among these candidate traits and U(crit). These data suggest that many traits, interactions among traits and traits not measured in this study affect prolonged swimming performance in threespine stickleback.

Evolution of stickleback spines through independent cis-regulatory changes at HOXDB.

Understanding the mechanisms leading to new traits or additional features in organisms is a fundamental goal of evolutionary biology. We show that HOXDB regulatory changes have been used repeatedly in different fish genera to alter the length and number of the prominent dorsal spines used to classify stickleback species. In Gasterosteus aculeatus (typically 'three-spine sticklebacks'), a variant HOXDB allele is genetically linked to shortening an existing spine and adding an additional spine. In Apeltes quadracus (typically 'four-spine sticklebacks'), a variant HOXDB allele is associated with lengthening a spine and adding an additional spine in natural populations. The variant alleles alter the same non-coding enhancer region in the HOXDB locus but do so by diverse mechanisms, including single-nucleotide polymorphisms, deletions and transposable element insertions. The independent regulatory changes are linked to anterior expansion or contraction of HOXDB expression. We propose that associated changes in spine lengths and numbers are partial identity transformations in a repeating skeletal series that forms major defensive structures in fish. Our findings support the long-standing hypothesis that natural Hox gene variation underlies key patterning changes in wild populations and illustrate how different mutational mechanisms affecting the same region may produce opposite gene expression changes with similar phenotypic outcomes.

Using asexual vertebrates to study genome evolution and animal physiology: Banded (Fundulus diaphanus) x Common Killifish (F. heteroclitus) hybrid lineages as a model system.

Wild, asexual, vertebrate hybrids have many characteristics that make them good model systems for studying how genomes evolve and epigenetic modifications influence animal physiology. In particular, the formation of asexual hybrid lineages is a form of reproductive incompatibility, but we know little about the genetic and genomic mechanisms by which this mode of reproductive isolation proceeds in animals. Asexual lineages also provide researchers with the ability to produce genetically identical individuals, enabling the study of autonomous epigenetic modifications without the confounds of genetic variation. Here, we briefly review the cellular and molecular mechanisms leading to asexual reproduction in vertebrates and the known genetic and epigenetic consequences of the loss of sex. We then specifically discuss what is known about asexual lineages of Fundulus diaphanus x F. heteroclitus to highlight gaps in our knowledge of the biology of these clones. Our preliminary studies of F. diaphanus and F. heteroclitus karyotypes from Porter's Lake (Nova Scotia, Canada) agree with data from other populations, suggesting a conserved interspecific chromosomal arrangement. In addition, genetic analyses suggest that: (a) the same major clonal lineage (Clone A) of F. diaphanus x F. heteroclitus has remained dominant over the past decade, (b) some minor clones have also persisted, (c) new clones may have recently formed, and iv) wild clones still mainly descend from F. diaphanus ♀ x F. heteroclitus ♂ crosses (96% in 2017-2018). These data suggest that clone formation may be a relatively rare, but continuous process, and there are persistent environmental or genetic factors causing a bias in cross direction. We end by describing our current research on the genomic causes and consequences of a transition to asexuality and the potential physiological consequences of epigenetic variation.

Linking genotypes to phenotypes and fitness: how mechanistic biology can inform molecular ecology.

The accessibility of new genomic resources, high-throughput molecular technologies and analytical approaches such as genome scans have made finding genes contributing to fitness variation in natural populations an increasingly feasible task. Once candidate genes are identified, we argue that it is necessary to take a mechanistic approach and work up through the levels of biological organization to fully understand the impacts of genetic variation at these candidate genes. We demonstrate how this approach provides testable hypotheses about the causal links among levels of biological organization, and assists in designing relevant experiments to test the effects of genetic variation on phenotype, whole-organism performance capabilities and fitness. We review some of the research programs that have incorporated mechanistic approaches when examining naturally occurring genetic and phenotypic variation and use these examples to highlight the value of developing a comprehensive understanding of the relationship between genotype and fitness. We give suggestions to guide future research aimed at uncovering and understanding the genetic basis of adaptation and argue that further integration of mechanistic approaches will help molecular ecologists better understand the evolution of natural populations.

Do differences in the activities of carbohydrate metabolism enzymes between Lake Whitefish ecotypes match predictions from transcriptomic studies?

Transcriptomic studies are facilitating the search for the molecular bases of adaptation in natural populations, but the impact of these differences in mRNA content on animal physiology are often unknown. One way to determine if molecular changes have the potential to influence animal physiology and performance is to test for correlated changes at higher levels of biological organization, including enzyme activity. Here, we measure the activities of carbohydrate metabolism enzymes to test if previously documented genetic and transcriptomic variation between 'dwarf' and 'normal' Lake Whitefish ecotypes are associated with corresponding changes in enzyme activity (measured as maximal rate, Vmax) in liver and skeletal muscle. We use laboratory-reared fish from the same populations as prior transcriptomic studies and find that white muscle mRNA content is a good predictor of glycolytic and glycogen metabolism enzyme activity, and dwarf whitefish have evolved higher activities than normal whitefish. However, the differences in hepatic mRNA content found between ecotypes in prior studies are not associated with comparable changes in enzyme activity. For example, dwarf whitefish have lower enzyme activities, but higher transcript abundances for two glycolytic enzymes compared to normal whitefish. Overall, we find that transcriptomic studies successfully highlight evolutionary variation in enzyme activities, but not always in the direction predicted, indicating that a variety of tissue-specific regulatory mechanisms contributed to the evolution of energy metabolism in Lake Whitefish.

Cline coupling and uncoupling in a stickleback hybrid zone.

Strong ecological selection on a genetic locus can maintain allele frequency differences between populations in different environments, even in the face of hybridization. When alleles at divergent loci come into tight linkage disequilibrium, selection acts on them as a unit and can significantly reduce gene flow. For populations interbreeding across a hybrid zone, linkage disequilibria between loci can force clines to share the same slopes and centers. However, strong ecological selection on a locus can also pull its cline away from the others, reducing linkage disequilibrium and weakening the barrier to gene flow. We looked for this "cline uncoupling" effect in a hybrid zone between stream resident and anadromous sticklebacks at two genes known to be under divergent natural selection (Eda and ATP1a1) and five morphological traits that repeatedly evolve in freshwater stickleback. These clines were all steep and located together at the top of the estuary, such that we found no evidence for cline uncoupling. However, we did not observe the stepped shape normally associated with steep concordant clines. It thus remains possible that these clines cluster together because their individual selection regimes are identical, but this would be very surprising given their diverse roles in osmoregulation, body armor, and swimming performance.

Adaptation and acclimation of aerobic exercise physiology in Lake Whitefish ecotypes (Coregonus clupeaformis).

The physiological mechanisms underlying local adaptation in natural populations of animals, and whether the same mechanisms contribute to adaptation and acclimation, are largely unknown. Therefore, we tested for evolutionary divergence in aerobic exercise physiology in laboratory bred, size-matched crosses of ancestral, benthic, normal Lake Whitefish (Coregonus clupeaformis) and derived, limnetic, more actively swimming "dwarf" ecotypes. We acclimated fish to constant swimming (emulating limnetic foraging) and control conditions (emulating normal activity levels) to simultaneously study phenotypic plasticity. We found extensive divergence between ecotypes: dwarf fish generally had constitutively higher values of traits related to oxygen transport (ventricle size) and use by skeletal muscle (percent oxidative muscle, mitochondrial content), and also evolved differential plasticity of mitochondrial function (Complex I activity and flux through Complexes I-IV and IV). The effects of swim training were less pronounced than differences among ecotypes and the traits which had a significant training effect (ventricle protein content, ventricle malate dehydrogenase activity, and muscle Complex V activity) did not differ among ecotypes. Only one trait, ventricle mass, varied in a similar manner with acclimation and adaptation and followed a pattern consistent with genetic accommodation. Overall, the physiological and biochemical mechanisms underlying acclimation and adaptation to swimming activity in Lake Whitefish differ.

RAD-QTL Mapping Reveals Both Genome-Level Parallelism and Different Genetic Architecture Underlying the Evolution of Body Shape in Lake Whitefish (Coregonus clupeaformis) Species Pairs.

Parallel changes in body shape may evolve in response to similar environmental conditions, but whether such parallel phenotypic changes share a common genetic basis is still debated. The goal of this study was to assess whether parallel phenotypic changes could be explained by genetic parallelism, multiple genetic routes, or both. We first provide evidence for parallelism in fish shape by using geometric morphometrics among 300 fish representing five species pairs of Lake Whitefish. Using a genetic map comprising 3438 restriction site-associated DNA sequencing single-nucleotide polymorphisms, we then identified quantitative trait loci underlying body shape traits in a backcross family reared in the laboratory. A total of 138 body shape quantitative trait loci were identified in this cross, thus revealing a highly polygenic architecture of body shape in Lake Whitefish. Third, we tested for evidence of genetic parallelism among independent wild populations using both a single-locus method (outlier analysis) and a polygenic approach (analysis of covariation among markers). The single-locus approach provided limited evidence for genetic parallelism. However, the polygenic analysis revealed genetic parallelism for three of the five lakes, which differed from the two other lakes. These results provide evidence for both genetic parallelism and multiple genetic routes underlying parallel phenotypic evolution in fish shape among populations occupying similar ecological niches.